The first activating TSH receptor mutation in transmembrane domain 1 identified in a family with nonautoimmune hyperthyroidism.

Sporadic and familial nonautoimmune hyperthyroidism are very rarely occurring diseases. Within the last years constitutively activating TSH receptor mutations were identified as one possible pathomechanism. Except for S281N in the extracellular N-terminal domain, all other germline mutations are located in the transmembrane domains 2, 3, 5, 6, and 7 of the TSH receptor, whereas no mutation was reported in transmembrane domains 1 and 4 to date. Here we report the first family with a constitutively active TSHR mutation in transmembrane domain 1 resulting in a substitution of the conserved Gly(431) for Ser. This mutation was found in the investigated patient, his father, and the paternal grandmother. As known from other familial cases of nonautoimmune hyperthyroidism, the age of onset of the disease was variable, ranging from early childhood in the patient and his father to adolescence in the grandmother. Functional characterization of this mutation showed a constitutive activation of the G(s)/adenylyl cyclase system. Moreover, this germline mutation also activates the G(q/11)/phospholipase C pathway. The importance of Gly(431) for receptor quiescence is supported further by introduction of other mutations at this position, all leading to constitutive receptor activity. Our data show now that constitutively activating mutations can be found in the entire transmembrane domain region of the TSH receptor, indicating the important role of all parts of the transmembrane domain region for maintaining the inactive receptor conformation.

A variation of vitamin D deficiency in children.

We report three children presenting with hypocalcemia, hyperphosphatemia, elevated levels of parathyroid hormone, low concentrations of 25(OH)-vitamin D, normal to elevated concentrations of 1,25(OH)2-vitamin D, and normal radiographs. Although these findings led to consideration of parathyroid hormone resistance, clinical and biochemical findings remained normal after discontinuation of therapy, suggesting a variation of vitamin D deficiency.

Cardiovascular risk factors in young adult survivors of childhood acute lymphoblastic leukemia.

PURPOSE: To assess cardiovascular risk factors (CVRF) in young adult survivors of childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Twenty-six subjects (median age, 20.9 years; median interval since completion of therapy, 13.3 years) were evaluated. Ten participants had received cranial irradiation (CRT), whereas 16 had received only chemotherapy. Primary outcome measures included body mass index (BMI), blood pressure, fasting lipoprotein, glucose, and insulin levels. Secondary measures included insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 levels, physical activity index, a 7-day dietary recall, tobacco product use, and measurement of the intima-media thickness (IMT) of the common carotid artery. RESULTS: Sixty-two percent (16/26) of participants had at least one CVRF potentially related to their cancer treatment (obesity, dyslipidemia, increased blood pressure, or insulin resistance), with 30% (7/26) having more than two CVRF. Thirty-one percent (8/26) of subjects were obese (BMI > or = 30). Subjects who were treated with CRT (BMI, 30.4 +/- 6.7) had an increased BMI (P = 0.039) in comparison with those who received only chemotherapy (BMI, 25.4 +/- 5.1). Triglyceride and very low-density lipoprotein C levels were significantly higher in those treated with CRT (P = 0.027 and 0.022, respectively). The IGF-1 was inversely correlated with IMT (total group, -0.514, P = 0.009; females only, -0.729, P = 0.003). CONCLUSIONS: Young adult survivors of childhood ALL, especially those treated with CRT, are at risk for obesity and dyslipidemia, insulin resistance, hypertension, and cardiovascular disease. Further investigation of these risks is warranted.

Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures.

The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

Upregulation of gap junctional intercellular communication in immortalized gonadotropin-releasing hormone neurons by stimulation of the cyclic AMP pathway.

Increased gap junctional intercellular communication induced by agents that stimulate the adenylyl cyclase/cAMP pathway was observed in the GnRH-secreting neuronal cell line, GT1-7, and possible underlying mechanisms were examined. A 24-hour treatment of GT1-7 neurons with 100 microM dibutyryl cAMP + 100 microM IBMX or with 2 microM forskolin increased by greater than 2-fold the percentage of cells that were dye coupled, using the noninvasive dye coupling assay, fluorescent recovery after photobleaching (FRAP). Longer treatment times (48 h) and higher concentrations of dibutyryl cAMP (500 microM) did not further increase the percentage of dye-coupled cells, while there was no increase in dye coupling observed between untreated cells and cells treated for 2 h or less. The increase in dye coupling induced by dibutyryl cAMP/IBMX was inhibited by octanol or dieldrin, agents known to block gap junction-mediated intercellular coupling in other cell types. Western blot analysis of total protein or membrane protein-enriched extracts revealed no apparent difference in the cellular levels of connexin 26, a connexin subtype previously shown to be expressed by GT1-7 cells, between untreated cells and cells treated for 24 h with dibutyryl cAMP/IBMX or forskolin. In addition, expression of connexin 32 or 43 protein before or after treatment was not detected. On the other hand, a dramatic increase in both the number of neurites and neurites that immunostained positive for connexin 26 was observed in dibutyryl cAMP/IBMX-treated cells. We hypothesize that the observed increase in dye coupling between GT1-7 neurons following stimulation of the adenylyl cyclase/cAMP pathway results from an augmentation of cell-cell contacts due to an increased number of neurites containing gap junctional plaques, possibly through an effect on cellular differentiation.

Growth hormone therapy in children with short stature: is bigger better or achievable?

The introduction of recombinant DNA-synthesized human growth hormone in the mid-1980s, and its attendant unlimited supply, have led to wider application of growth hormone therapy in children. Over the past decade, the efficacy of growth hormone treatment in patients with Turner syndrome and chronic renal insufficiency, two conditions in which growth hormone secretion is normal, in improving growth velocity and final height, has also led to the consideration of growth hormone therapy in children with idiopathic short stature. Although thousands of patients with idiopathic short stature are currently being treated with growth hormone, the limited overall results available at this time do not show a significant improvement in final adult height despite an improvement in short-term growth velocity. Potential reasons for this outcome include 1) skeletal age advancing more rapidly than height age, 2) heterogeneity of the patient population comprising idiopathic short stature, 3) inherent inaccuracies of methodological tools, such as measurement of predicted adult height, and 4) a subset of children with idiopathic short stature who may, in fact, have partial growth hormone insensitivity. From a psychological perspective, the consensus of investigations in non-clinic-referred populations of psychosocial function in children with short stature do not indicate a disadvantage compared with children of normal height when socio-economic status is taken into consideration. These results, in conjunction with the minimal gains reported in behavioural measurements in idiopathic short children treated with growth hormone, question the traditional rationale that augmentation of growth velocity results in improvement in psychosocial well-being.

Insulin syringe disposal practices of pediatric patients with insulin-dependent diabetes mellitus.

Pediatric patients with insulin-dependent diabetes mellitus were surveyed over a 5-month period to determine disposal practices of insulin syringes. Eighty-nine (79.5%) of 112 patients surveyed responded. Thirty-three percent of the responders disposed of syringes in accordance with recommendations of the local health department and the American Diabetes Association, while the remaining patients did not. Fifty-two percent of the patients rendered the syringes useless before disposal. The study revealed a need to educate and inform patients on the proper procedures that should be followed when disposing of syringes in the home.

Immortalized hypothalamic luteinizing hormone-releasing hormone neurons express a connexin 26-like protein and display functional gap junction coupling assayed by fluorescence recovery after photobleaching.

Expression of gap junction proteins was studied in the LHRH neuronal cell line, GT1-7, as a first step in defining the signalling mechanisms responsible for the pulsatile secretion of LHRH. GT1-7 cells were found to express a connexin 26-like protein that comigrated with mouse liver connexin 26 and that reacted with connexin 26-specific antibodies on Western blots. Immunofluorescent staining revealed punctate staining in a fraction of the cells, often present at points of apparent contact with neighboring cell bodies or processes. Fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein loaded GT1-7 cells showed dye coupling among 20-30% of cells that made contact with other cells, suggesting the presence of functional gap junctions in this cell line.

Longitudinal assessment of L-thyroxine therapy for congenital hypothyroidism.

We evaluated the longitudinal response in 43 infants with congenital primary hypothyroidism during the first year of L-thyroxine therapy. Diagnosis was confirmed by serum thyroid hormone measurements by 4 weeks of age in 38 infants and between 40 and 80 days of age in the remainder. This group of infants was divided by radionuclide thyroid imaging into 34 infants with thyroid dysgenesis and nine with dyshormonogenesis. The group with thyroid dysgenesis was subdivided into 21 infants with athyreosis and 13 with residual thyroid tissue (11 ectopic and 2 hypoplastic glands). L-Thyroxine therapy, at an average dose of 10 to 14 micrograms/kg/day, was begun immediately after diagnosis, and serum concentration of total thyroxine, free thyroxine, triiodothyronine, reverse triiodothyronine, and thyroid-stimulating hormone were determined serially. Serum concentration of total and of free thyroxine became normal within 1 week of the start of therapy in all groups. Despite a similarly mild degree of hypothyroidism at diagnosis observed in infants with dyshormonogenesis or with ectopia or hypoplasia, those with dyshormonogenesis had a more sensitive response to initial thyroid hormone replacement than did patients with thyroid dysgenesis, as judged by L-thyroxine does and thyroid-stimulating hormone suppression. We conclude that the prompt restoration of clinical and biochemical euthyroidism during early infancy with doses of L-thyroxine between 10 and 14 micrograms/kg/day is a safe and effective method of therapy for children with congenital hypothyroidism.

Hypophysiotropic signal frequency and the functioning of the pituitary-ovarian system in the rhesus monkey.

In adult rhesus monkeys bearing hypothalamic lesions that abolished secretions of LH and FSH, normal circulating levels of these hormones and consequent follicular development culminating in ovulation were reestablished by the administration of a GnRH pulse once every hour (control frequency). We examined the effects of slowing the frequency of these pulses on ovarian follicular development, as assessed by circulating concentrations of estradiol. A reduction in frequency to one pulse every 90 min supported follicular development in most instances, albeit with a diminished incidence of ovulation. One pulse of GnRH every 2 h resulted in anovulatory follicular cycles with lower peak concentrations of estradiol than those achieved on the control frequency. Follicular development was absent when GnRH pulses were delivered once every 3 h. When mean concentrations of gonadotropins were determined during periods of low circulating estradiol levels, plasma FSH concentrations in monkeys receiving GnRH pulses at the slow rates did not differ from control, whereas LH concentrations were significantly reduced. It can be concluded that small reductions in the frequency of GnRH stimulation have profound effects on the quality of follicular development, even when FSH concentrations are maintained at normal levels.